Figure 2

PTPRRexpression is an early and directly repressed target of the AR. (A) LNCaP cells were cultured in medium supplemented with 10% dextran charcoal stripped FBS to produce a steroid deplete medium. Following culture for 72 hours, cells were treated with 10 nM synthetic androgen analogue methyltrienolone (R1881) for the times indicated. (A) Expression of PTPRR mRNA in cells grown in steroid deplete (SD) or androgen (A+) treated conditions over a 24 hours period. The response to androgens was confirmed using PSA (KLK3) expression (not shown). (B) Expression of PTPRR protein is reduced in LNCaP cells treated with 10 nM R1881 for 24 and 48 hours as detected by western blot. Actin was used as a loading control. (C) Repression of PTPRR is also evident in LNCaP cells treated with 0.1 to 100 nM of R1881. Relative PTPRR expression was detected by real-time PCR. (D) The reduction in PTPRR mRNA expression in response to androgens is still seen in the presence of 1 μg/ml cycloheximide (CHX) as confirmed by real-time PCR. (E) Repression of PTPRR protein expression by the AR is inhibited in the presence of 10 μM of the anti-androgen Casodex (bicalutamide) (lane 6) and by 10 μM of flutamide (F). (G) Depletion of AR protein in LNCaP cells by esiRNA shows that when the AR is depleted there is no reduction in PTPRR protein in response to androgens. (H) Immunofluorescent staining of LNCaP cells grown in steroid depleted conditions indicates that PTPRR is localised to the cytoplasm. Bar is 10 μM. The specificity of the antisera was confirmed with pre-absorption with the immunising peptide, and by detection of over-expressed protein and esiRNA mediated protein depletion (Additional file 3: Figure S2, Figure 3A and 3D). (I) The PTPRR gene and protein structure is illustrated. The gene consists of 14 exons, and codes for a 74 kDa protein which contains a signal peptide (SD domain), a transmembrane domain (TM) and a kinase interaction motif (KIM).